Superoxide generation contributes to osteoclastic bone resorption. White cells generate superoxide by activating NADPH oxidase, a membrane-bound enzyme, which results in generation of superoxide on the non-cytoplasmic side of the cellular membranes. Preliminary data suggest that osteoclastic superoxide production can be inhibited by an NADPH oxidase inhibitor. Thus, the central hypothesis proposes that NADPH oxidase generates superoxide at the osteoclastic-bone interface and this superoxide contributes directly to degradation of bone matrix proteins. Three lines of investigation will be followed. First, the applicants plan to determine if inhibition of NADPH oxidase by an iodonium compound, diphenylene iondonium significantly reduces the production of superoxide in osteoclasts during unstimulated and stimulated bone resorption. These experiments will use calvarial explants from normal and osteopetrotic mice. The microphthalmic mouse model has provided strong evidence that increased superoxide production can stimulate osteoclastic bone resorption. Diphenylene iondonium, a specific NADPH oxidase inhibitor, will be used to determine if there is a correlation between the rates of bone resorption and osteoclastic superoxide generation. Second, the subcellular localization of NADPH oxidase in mouse osteoclasts will be determined using antibodies specific for subunits of cytochrome b558, a major component of the NADPH oxidase enzyme. These studies will determine if the immunocytochemical location of NADPH oxidase correlates with areas of superoxide production, measured by electron microscopic visualization of diformazan granules. Finally, at least three mechanisms of increasing NADPH oxidase exist in white cells. These include: (a) an increase in the total amount; (b) a change in the subcellular distribution; and (c) a change in the state of activation of existing enzyme. Mapping out the subcellular localization of NADPH oxidase and the corresponding areas of superoxide production should contribute insight into the role of this enzyme in modulating osteoclastic bone resorptive activity. Delineation of a specific enzyme(s) responsible for superoxide formation and identifying the mechanisms leading to alterations in the function of these enzymes will provide new insight into the defect(s) in animals and humans with osteopetrosis and osteoporosis. Successful completion of these studies should pave the way to improved treatment strategies for treating osteopetrosis with stimulators of NADPH oxidase and osteoporosis, with inhibitors.